Publications (6)34.23 Total impact
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Article: Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance.
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ABSTRACT: The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives), together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses, Affymetrix profiling, real-time PCR, and immunoprecipitation. Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g., POU5F1, NANOG, SOX2, FOXD3). siRNA analyses identified two retinoblastoma binding proteins (RBBP4, RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells, and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1, NANOG, SOX2, and/or FOXD3 plus certain cell cycle genes (e.g., CCNA2, CCNB1), while increasing expression of genes involved in organogenesis (particularly neurogenesis). These results reveal new candidate positive regulators of human PS cells, providing evidence of their ability to regulate expression of pluripotency, cell cycle, and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency.Experimental hematology 05/2011; 39(8):866-79.e1. · 3.11 Impact Factor -
Article: Functional assays for human embryonic stem cell pluripotency.
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ABSTRACT: Realizing the potential that human embryonic stem cells (hESCs) hold, both for the advancement of biomedical science and the development of new treatments for many human disorders, will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard, it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay, thus, provides a valuable tool for monitoring the quality of hESC cultures, and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies), or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm, mesoderm, and ectoderm lineages.Methods in molecular biology (Clifton, N.J.) 01/2011; 690:67-80. -
Article: Enhanced generation of hematopoietic cells from human hepatocarcinoma cell-stimulated human embryonic and induced pluripotent stem cells.
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ABSTRACT: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) constitute unique sources of pluripotent cells, although the molecular mechanisms involved in their differentiation into specific lineages are just beginning to be defined. Here we evaluated the ability of MEDII (medium conditioned by HepG2 cells, a human hepatocarcinoma cell line) to selectively enhance generation of mesodermal derivatives, including hematopoietic cells, from hESCs and hiPSCs. Test cells were exposed to MEDII prior to being placed in conditions that promote embryoid body (EB) formation. Hematopoietic activity was measured by clonogenic assays, flow cytometry, quantitative real-time polymerase chain reaction of specific transcript complementary DNAs and the ability of cells to repopulate sublethally irradiated nonobese diabetic/severe combined immunodeficient interleukin-2 receptor gamma-chain-null mice for almost 1 year. Exposure of both hESCs and hiPSCs to MEDII induced a rapid and preferential differentiation of hESCs into mesodermal elements. Subsequently produced EBs showed a further enhanced expression of transcripts characteristic of multiple mesodermal lineages, and a concurrent decrease in endodermal and ectodermal cell transcripts. Frequency of all types of clonogenic hematopoietic progenitors in subsequently derived EBs was also increased. In vivo assays of MEDII-treated hESC-derived EBs also showed they contained cells able to undertake low-level but longterm multilineage repopulation of primary and secondary nonobese diabetic/severe combined immunodeficient interleukin-2 receptor gamma-chain-null mice. MEDII treatment of hESCs and hiPSCs alike selectively enhances their differentiation into mesodermal cells and allows subsequent generation of detectable levels of hematopoietic progenitors with in vitro and in vivo differentiating activity.Experimental hematology 06/2009; 37(8):924-36. · 3.11 Impact Factor -
Article: Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells.
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ABSTRACT: Human embryonic stem cells (hESCs) can be maintained in vitro as immortal pluripotent cells but remain responsive to many differentiation-inducing signals. Investigation of the initial critical events involved in differentiation induction would be greatly facilitated if a specific, robust, and quantitative assay for pluripotent hESCs with self-renewal potential were available. Here we describe the results of a series of experiments to determine whether the formation of adherent alkaline phosphatase-positive (AP(+)) colonies under conditions optimized for propagating undifferentiated hESCs would meet this need. The findings can be summarized as follows. (a) Most colonies obtained under these conditions consist of >or=30 AP(+) cells that coexpress OCT4, NANOG, SSEA3, SSEA4, TRA-1-60, and TRA-1-81. (b) Most such colonies are derived from SSEA3(+) cells. (c) Primary colonies contain cells that produce secondary colonies of the same composition, including cells that initiate multilineage differentiation in embryoid bodies (EBs). (d) Colony formation is independent of plating density or the colony-forming cell (CFC) content of the test population over a wide range of cell concentrations. (e) CFC frequencies decrease when differentiation is induced by exposure either to retinoic acid or to conditions that stimulate EB formation. Interestingly, this loss of AP(+) clonogenic potential also occurs more rapidly than the loss of SSEA3 or OCT4 expression. The CFC assay thus provides a simple, reliable, broadly applicable, and highly specific functional assay for quantifying undifferentiated hESCs with self-renewal potential. Its use under standardized assay conditions should enhance future elucidation of the mechanisms that regulate hESC propagation and their early differentiation.Stem Cells 06/2008; 26(5):1109-16. · 7.78 Impact Factor -
Article: Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells.
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ABSTRACT: MicroRNAs (miRNAs) are emerging as important, albeit poorly characterized, regulators of biological processes. Key to further elucidation of their roles is the generation of more complete lists of their numbers and expression changes in different cell states. Here, we report a new method for surveying the expression of small RNAs, including microRNAs, using Illumina sequencing technology. We also present a set of methods for annotating sequences deriving from known miRNAs, identifying variability in mature miRNA sequences, and identifying sequences belonging to previously unidentified miRNA genes. Application of this approach to RNA from human embryonic stem cells obtained before and after their differentiation into embryoid bodies revealed the sequences and expression levels of 334 known plus 104 novel miRNA genes. One hundred seventy-one known and 23 novel microRNA sequences exhibited significant expression differences between these two developmental states. Owing to the increased number of sequence reads, these libraries represent the deepest miRNA sampling to date, spanning nearly six orders of magnitude of expression. The predicted targets of those miRNAs enriched in either sample shared common features. Included among the high-ranked predicted gene targets are those implicated in differentiation, cell cycle control, programmed cell death, and transcriptional regulation.Genome Research 05/2008; 18(4):610-21. · 13.61 Impact Factor -
Article: LongSAGE profiling of nine human embryonic stem cell lines.
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ABSTRACT: To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.Genome biology 02/2007; 8(6):R113. · 6.63 Impact Factor
Top co-authors
Top Journals
Institutions
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2011
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University of Western Sydney
Penrith, New South Wales, Australia
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2009–2011
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Terry Fox Laboratory
Vancouver, British Columbia, Canada
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2008
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BC Cancer Research Centre
- Terry Fox Laboratory
Vancouver, British Columbia, Canada
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